Thesis (MSc (Biomedical Sciences))--Cape Peninsula University of Technology, 2018. / Background: Early detection of individuals who are at risk of developing Glucose Intolerance would decrease the morbidity and mortality associated with this disease. MicroRNA is one of the most widely studied biomolecules involved in epigenetic mechanisms, hence it offers unique opportunities in this regard. Circulating microRNAs are associated with disease pathogenesis during the asymptomatic stage of disease. This has therefore attracted a lot of attention as a potential biomarker for identifying individuals who have an increased risk of developing Glucose Intolerance. The identification of high risk biomarkers for Glucose Intolerance will go a long way to eliminate the possible complications that arise due to late diagnosis and treatment of Glucose Intolerance. This could ultimately lead to better ways to prevent, manage and control the Glucose Intolerance epidemic that is rampant worldwide. The aim of the study is to investigate expression of circulating microRNA’s in blood of mixed ancestry subjects with glucose intolerance. Methods: A quantitative cross-sectional study design involving 36 individuals [who were age, gender and BMI (Body Mass Index) matched] from a total population of 1989 participants of mixed ancestry descent, residing in Bellville South, South Africa was used. Participants were classified as controls (normoglycemic), pre-diabetic (preDM) and diabetic (DM) (screen detected diabetic) according to WHO criteria of 1998. MicroRNAs were extracted from serum using the Qiagen miRNeasy Serum/Plasma Kit (ThermoFisher). The purified micro RNAs were reverse-transcribed to cDNA (complementary deoxyribonucleic acid) using the Qiagen RT2 First Strand Kit. Then, using Qiagen miScript SYBR Green PCR kit and miScript miRNA PCR arrays (ThermoFisher), the real time polymerase chain reaction was done to determine the expression profile the circulating micro RNAs present in the serum of the participants. Results: The 36 participants were evenly divided into 3 groups of 12 participants each as mentioned earlier. There were significant differences between groups in the waist (cm) (p=0.0415) and waist/hip ratio (p=0.0011) with highest values in the DM group and lowest in the normal group. Clinical parameters varied significantly according to glycemic status. As expected, the FBG (mmol/L) (p<0.0001), 2 HRs Post Glucose (mmol/L) (p<0.0001), HbA1c (%) (p=0.0009), Fasting Insulin (mIU/L) (p=0.0039), were all highest in the DM and lowest in the control group. In contrast, the 2 HRs Post Insulin (mIU/L) (p = 0.0027) was highest in the preDM group and lowest in the normal group, while the Glucose/Insulin ratio (p=0.0477) was highest in the normal group and lowest in the preDM group. Triglycerides (mmol/L) (p=0.0043) and Total Chol (mmol/L) (p=0.0429) were significantly increased through the three groups, with highest values in the DM group and lowest in the normal group. Furthermore, 12 of the 84 miRNAs studied were expressed through all the 3 groups and they exhibited both inverse and positive correlations between the clinical parameters, especially the glucose parameters (Fasting blood glucose, 2 hours post glucose, Fasting blood insulin, 2 hours post insulin and Glycated Hemoglobin).

Chan Chung Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 93-101). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ix / List of Abbreviations --- p.xvii / Chapter CHATPER ONE --- INTRODUCTION / Chapter 1.1 --- Glucose Homeostasis --- p.1 / Chapter 1.1.1 --- Function --- p.1 / Chapter 1.1.2 --- Origins and regulation of glucose --- p.2 / Chapter 1.1.3 --- Glucoregulatory factors --- p.4 / Chapter 1.1.4 --- Insulin --- p.6 / Chapter 1.1.4.1 --- Function of Insulin --- p.7 / Chapter 1.1.4.2 --- Discovery and Production of Insulin --- p.7 / Chapter 1.1.4.3 --- Insulin Signaling Pathway --- p.8 / Chapter 1.1.4.3.1 --- Insulin Receptor --- p.8 / Chapter 1.1.4.3.2 --- MAPK Pathway --- p.9 / Chapter 1.1.4.3.3 --- Phosphatidylinositol 3-kinase (PI3-K) Pathway --- p.10 / Chapter 1.1.5 --- Glucose Transporters --- p.11 / Chapter 1.1.6 --- Role of skeletal muscle in glucose homeostasis --- p.13 / Chapter 1.1.7 --- Insulin Resistance --- p.14 / Chapter 1.1.8 --- Glucose abnormality and its complications --- p.16 / Chapter 1.2 --- Actin --- p.19 / Chapter 1.2.1 --- Function of Actin --- p.20 / Chapter 1.2.2 --- Actin Accessory Protein --- p.22 / Chapter 1.2.3 --- Actin Polymerization --- p.23 / Chapter 1.3 --- "Interaction between Insulin, GLUT4 and Actin in Glucose Homeostasis" --- p.24 / Chapter 1.3.1 --- Insulin-Induced Actin Remodeling --- p.25 / Chapter 1.3.2 --- Actin Remodeling and Insulin-Induced GLUT4 Translocation --- p.26 / Chapter 1.3.3 --- Involvement of Insulin Signaling Molecules in Actin Remodeling --- p.27 / Chapter 1.3.4 --- Actin Remodeling and Insulin Resistance --- p.30 / Chapter 1.4 --- Hypothesis and Objective --- p.30 / Chapter 1.4.1 --- Rationale --- p.30 / Chapter 1.4.2 --- Hypothesis --- p.31 / Chapter 1.4.3 --- Objective --- p.31 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Cell Culture --- p.36 / Chapter 2.2.1 --- Cell Culture --- p.36 / Chapter 2.2.2 --- Reagents Preparation and Incubation --- p.39 / Chapter 2.3 --- 2-Deoxyglucose Uptake --- p.39 / Chapter 2.4 --- Immunofluorescence Microscopy --- p.41 / Chapter 2.4.1 --- Permeabilized cell staining --- p.41 / Chapter 2.4.2 --- Membrane-intact cell staining --- p.43 / Chapter 2.4.3 --- The analysis of actin remodeling reduction --- p.44 / Chapter 2.5 --- Live Image Microscopy --- p.44 / Chapter 2.6 --- Transmission Electron Microscope Study --- p.44 / Chapter 2.7 --- Statistical Analysis --- p.46 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Cell Growth --- p.48 / Chapter 3.2 --- Acute Effect of Insulin on L6 myotubes --- p.48 / Chapter 3.2.1 --- Immunofluorescence Microscopy --- p.49 / Chapter 3.2.1.1 --- The time profile of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.49 / Chapter 3.2.1.2 --- The concentration effect of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.50 / Chapter 3.2.1.3 --- Relationship between actin cytoskeleton and GLUT4mycin permeabilized L6 myotubes --- p.51 / Chapter 3.2.1.4 --- Translocation of GLUT4myc in membrane-intact L6 myotubes --- p.51 / Chapter 3.2.1.5 --- "Effect of methyl-β-cyclodextrins, MeOH or EtOHin permeabilized and membrane-intact L6 myotubes" --- p.52 / Chapter 3.2.2 --- 2-Deoxyglucose Uptake --- p.52 / Chapter 3.2.2.1 --- "Effects of insulin, methyl-β-cyclodextrins, MeOH and EtOH in L6 myotubes" --- p.52 / Chapter 3.2.3 --- TEM Study --- p.53 / Chapter 3.2.3.1 --- Effects of insulin on actin cytoskeleton and GLUT4myc in L6 myotubes --- p.53 / Chapter 3.3 --- Effect of high glucose and high insulin incubation in L6 myotubes --- p.54 / Chapter 3.3.1 --- Immunofluorescence Microscopy --- p.54 / Chapter 3.3.1.1 --- High insulin and high glucose preincubation in permeabilized L6 myotubes --- p.55 / Chapter 3.3.1.2 --- Effect of high insulin and high glucose incubationin membrane-intact L6 myotubes --- p.55 / Chapter 3.3.2 --- 2-Deoxyglucose Uptake --- p.56 / Chapter 3.3.2.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.56 / Chapter 3.3.3 --- TEM Study --- p.57 / Chapter 3.3.3.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.57 / Chapter 3.4 --- Effect of FFA incubation in L6 myotubes --- p.58 / Chapter 3.4.1 --- Immunofluorescence Microscopy --- p.58 / Chapter 3.4.1.1 --- FFA preincubation in permeabilized L6 myotubes --- p.58 / Chapter 3.4.1.2 --- FFA incubation in membrane-intact L6 myotubes --- p.59 / Chapter 3.4.2 --- 2-Deoxyglucose Uptake --- p.59 / Chapter 3.4.2.1 --- FFA incubation in L6 myotubes (24 hours) --- p.60 / Chapter 3.4.3 --- TEM Study --- p.62 / Chapter 3.4.3.1 --- FFA incubation in L6 myotubes --- p.62 / Chapter 3.5 --- Effect of CHO incubation in L6 myotubes --- p.62 / Chapter 3.5.1 --- Immunofluorescence Microscopy --- p.62 / Chapter 3.5.1.1 --- CHO preincubation in permeabilized L6 myotubes --- p.63 / Chapter 3.5.1.2 --- CHO incubation in membrane-intact L6 myotubes --- p.63 / Chapter 3.5.2 --- 2-Deoxyglucose Uptake --- p.64 / Chapter 3.5.2.1 --- CHO incubation in L6 myotubes (24 hours) --- p.64 / Chapter 3.5.3 --- TEM Study --- p.65 / Chapter 3.5.3.1 --- CHO incubation in L6 myotubes --- p.65 / Chapter 3.6 --- Overall changes in glucose uptake after preincubation experiment --- p.65 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Effect of insulin on L6 myotubes --- p.69 / Chapter 4.2 --- "Effect of methyl-β-cyclodextrins, MeOH and EtOH on L6 myotube" --- p.75 / Chapter 4.3 --- Effect of pretreatment of cells in conditions of insulin resistance --- p.76 / Chapter 4.3.1 --- Effect of high glucose and high insulin preincubation on L6 myotubes --- p.76 / Chapter 4.3.2 --- Effect of FFA preincubation on L6 myotubes --- p.78 / Chapter 4.3.3 --- Effect of CHO preincubation on L6 myotubes --- p.82 / Chapter 4.3.4 --- Effect of cell preincubation in conditions of insulin resistance on L6 myotubes (TEM) --- p.83 / Chapter 4.4 --- Summary of the effects of cell preincubation in conditions of insulin resistance --- p.84 / Chapter 4.5 --- Possible mechanisms involved in insulin resistance induction --- p.86 / Chapter 4.5.1 --- Possible changes in GLUT expression and activities --- p.87 / Chapter 4.5.2 --- Possible changes in insulin signaling propagation --- p.88 / Chapter 4.5.3 --- Altered functioning of various actin accessory proteins --- p.89 / Chapter 4.6 --- Limitation of the study --- p.90 / Chapter 4.7 --- Conclusion --- p.90 / Chapter 4.8 --- Future study --- p.91 / REFERENCES --- p.93 / TABLES

Photocopy of typescript. / Digitized by Kansas Correctional Industries

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that 13C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained to a remarkable extent during an ischemic time course. These findings suggested an unexpected source of glucose in HIF-1α hearts during global ischemia. Accordingly, the presence of gluconeogenesis in hearts was evaluated. Indeed, gluconeogenic intermediates (i.e. m+3) including glucose-6-phosphate [m+3], fructose-6-phosphate [m+3], and fructose 1,6-bisphosphate [m+3] were observed at significantly elevated levels in the ischemic HIF-1α heart. Collectively, these data establish the surprising finding that HIF-1α supports active gluconeogenesis in the heart during ischemia. As less is known regarding the effects of CTRP3 we first tested whether CTRP3 overexpression would protect the ischemic heart. Our data indicate that CTRP3 failed to confer ischemic tolerance in heart ex vivo. However,we were able to show that CTRP3 protected the liver from lipid-induced stress and prevented hepatic lipid accumulation. To further investigate the mechanisms of hepatic protective effect mediated by CTRP3, we identified the receptor and established that CTRP3 increases oxygen consumption in response to lipid overloaded. Lysosomal-associated membrane protein 1 (LAMP-1), In summary, these data indicate that targeted metabolic rearrangements within cardiomyocyte/hepatocyte holds promise for the alleviation of common pathological conditions.

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The effects of maternal dietary glucose restriction on reproductive performance were investigated by feeding pregnant rats isocaloric diets containing graded levels of dietary glucose (0, 12, 24 and 60%) during pregnancy and during pregnancy and lactation, and by measuring the effects of glucose restriction on (1) maternal, fetal and neonatal metabolism, on (2) growth and composition of the mammary glands and placentas, and (3) on milk composition. Carbohydrate restriction induced maternal metabolic adaptations that were proportional to the severity of the glucose restriction. Placental growth and composition as well as mammary gland composition were not affected by dietary glucose restriction, whereas fetal growth and development and milk composition were significantly impaired when glucose was limited in the maternal diet. This suggests that the effects of dietary glucose on the fetus and on milk composition were not mediated by changes in placenta and mammary gland DNA, protein or glycogen concentrations. Complete dietary glucose restriction significantly depressed fetal liver, lung and heart glycogen concentrations; repletion of the maternal diets with 12 and 24% glucose restored cardiac glycogen to normal but not fetal lung glycogen and liver glycogen. Pups born to dams fed a glucose-free diet failed to survive longer than 24 h postpartum and that was associated with the low levels of tissue glycogen at birth in these pups. At birth, lung and liver glycogen concentration of pups of the 12 and 24% glucose diets was similar to pups of the control diet despite the fact that these reserves were depressed in utero; and these pups efficiently corrected the transient hypoglycemia observed following parturition. The effects of glucose restriction on fetal liver glycogen were not reflected by similar changes in fetal plasma insulin, glucagon and glucose levels or in glycogen synthase and phosphorylase activities. Maternal dietary glucose was an important determinant

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Since glucose is an essential nutrient for normal fetal growth and development, the impact of reduced maternal dietary glucose supply, on amniotic fluid (amf) amino acid composition was investigated. Furthermore, this study investigated whether any resulting changes in the concentrations of amf amino acids could be predictive of fetal growth and metabolic status. Pregnant rat dams were fed isocaloric diets containing graded levels of dietary glucose (0, 12, 24 and 60%) and the amf amino acid content was analysed on gestational days (gd) 18.5 to 21.5. Carbohydrate restriction produced significant increases in the concentrations of amf isoleucine (on gd 21.5), tryptophan (on gd 18.5 and 21.5) and 3-methylhistidine (on gd 20.5 and 21.5). An interaction between diet and day of gestation modified amf taurine levels such that dams fed low carbohydrate diets showed significant increases in amf taurine as pregnancy progressed. Specific amf amino acids correlated with fetal growth parameters and fetal tissue glycogen reserves indicating the ability of amf composition to reflect fetal distress under conditions of compromised maternal nutritional status. A greater statistical predictability of amf constituents was obtained with fetal growth parameters than with fetal tissue glycogen reserves. These results suggest that amf amino acids are better predictors of fetal growth status than of fetal metabolic status.